Project Summary Alcohol use disorder is a chronically relapsing brain disease that is often precipitated by stress. The source of the stress can either be external, such as an environmental stressor acting upon the affected individual, and/or internal, with endogenous signals challenging the system for more alcohol. This K99/R00 application aims to study the neural circuits modulating long-term, intermittent alcohol (IA) drinking and stress. The dynorphin (DYN)/kappa opioid receptor neuropeptide system has been shown to be involved in the dysphoric phases of alcohol dependence in preclinical studies. The K99 portion of this proposal will involve more extensive training using cell-specific optogenetics and chemogenetics to influence alcohol-affected behavior. Specifically, this proposal tests the control of stress coping behavior in response to a predator odor during protracted withdrawal from alcohol in the bed nucleus of the stria terminalis (BNST), a brain area that has DYN adaptations following chronic IA drinking. Additional training will be gained during the second K99 phase using a discovery-based approach to identify whole brain circuit mapping after escalated alcohol drinking and stress. To probe which BNST inputs are recruited in an unbiased manner, an inducible cre-dependent retrograde virus will be used in fos-cre (Targeted Recombination of Active Populations, TRAP2) mice to identify activated inputs to the BNST that are time-locked to acute predator odor stress; these inputs may be differentially modulated by a history of IA drinking. Next, the TRAPed BNST circuitry will be identified through the iDISCO+ whole brain tissue clearing and immunolabeling method using light sheet microscopy and computational analysis aligned with the Allen Brain Atlas. Further, whole cell patch clamp recordings of DYN cells will be performed to describe the synaptic alterations in the stressed, alcohol-exposed BNST-projecting pathways. These K99 experiments will yield newly identified whole brain neural systems recruited after alcohol and stress as future avenues for independent studies for the R00 phase. The R00 studies will assess the contributions of distinct circuits using both in vivo optogenetics and a multiplexed DREADD approach to inhibit specific BNST projections. This research would thereby enhance our understanding of the functional circuitry of the brain and inform the development of targeted treatments for stress and alcohol disorders. The postdoctoral candidate, Dr. Lara Hwa, will use the K99/R00 career development award to master these modern neuroscience techniques at the University of North Carolina with her qualified mentoring team to become an expert neuroscientist in alcohol and stress interactions. She hopes to lead a strong research and teaching program at an R1 academic institution, poised to probe the complex relationship of alcohol and stress underlying pathological behavior through advanced neuroscience techniques.